GSM6730099: testis, control_scRNAseq; Mus musculus; RNA-Seq
Decapsulated testes were incubated in DMEM containing 0.75 mg/ml collagenase type IV (Gibco, 17104-019) for 10 mins at 37°C with occasional inversion. Equal volume of DMEM was added to the suspension of dissociated tubules before centrifuging at 400 xg for 5 mins at 4 °C. After removing the supernatant, the cell pellet was washed in DMEM and resuspended in 0.05% trypsin-EDTA (Gibco, 25200-056) for 10 mins incubation at room temperature. An equal volume of ice-cold DMEM+10%FBS was then added to the cell suspension before washing the pellet twice with media. The cell pellet was resuspended in DPBS (Sigma, D8537) containing 0.04% bovine serum albumin (sigma, A9647) and filtered through a 100 µm filter (Falcon, 22363549) then a 40 µm filter (Falcon, 352340) to generate a single cell suspension. Single cell RNA-seq libraries were constructed from whole dissociated testis samples using the Chromium Next GEM Single Cell 3' GEM kit v3.1 (10x Genomics PN-1000121) according to the manufacturer's instructions. 150 bp paired end